ARTICLE Received 15 Jan 2015 | Accepted 19 Mar 2015 | Published 28 Apr 2015
DOI: 10.1038/ncomms7980
OPEN
FGF1 and FGF19 reverse diabetes by suppression of the hypothalamic–pituitary–adrenal axis Rachel J. Perry1,2,3, Sangwon Lee2,4, Lie Ma2,4, Dongyan Zhang1, Joseph Schlessinger2,4 & Gerald I. Shulman1,2,3
Fibroblast growth factor-1 (FGF1) and FGF19 have been shown to improve glucose metabolism in diabetic rodents, but how this occurs is unknown. Here to investigate the mechanism of action of these growth factors, we perform intracerebroventricular (ICV) injections of recombinant FGF1 or FGF19 in an awake rat model of type 1 diabetes (T1D) and measure rates of whole-body lipolysis, hepatic acetyl CoA content, pyruvate carboxylase activity and hepatic glucose production. We show that ICV injection of FGF19 or FGF1 leads to a B60% reduction in hepatic glucose production, hepatic acetyl CoA content and whole-body lipolysis, which results from decreases in plasma ACTH and corticosterone concentrations. These effects are abrogated by an intra-arterial infusion of corticosterone. Taken together these studies identify suppression of the HPA axis and ensuing reductions in hepatic acetyl CoA content as a common mechanism responsible for mediating the acute, insulin-independent, glucose-lowering effects of FGF1 and FGF19 in rodents with poorly controlled T1D.
1 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 2 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 3 Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 4 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA. Correspondence and requests for materials should be addressed to G.I.S. (email: [email protected]).
NATURE COMMUNICATIONS | 6:6980 | DOI: 10.1038/ncomms7980 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7980
R
ecent studies have generated a great deal of interest in the insulin-independent glucose-lowering effects of fibroblast growth factor-19 (FGF19) and FGF1 in diabetic rodents and their potential application as novel anti-diabetic therapies1–4; however, little is known regarding the mechanism by which these factors decrease plasma glucose concentrations. FGF19 is an atypical FGF which together with FGF21 and FGF23 is designated an endocrine FGF. It is now well established that members of the FGF family of growth factors mediate their cellular responses by binding together and acting in concert with heparin sulfate proteoglycans to activate the four receptor tyrosine kinases designated FGFR1–4. The endocrine members of the FGF family, designated FGF19, -21 and -23, mediate their endocrine functions by binding selectively to FGFR1c, FGFR2c, FGFR3c or FGFR4 together with b-Klotho or a-Klotho, respectively, to mediate their multiple endocrine functions1–3. FGF19 has been proposed to play diverse physiological roles, modulating development, metabolism, neuronal signalling, atherosclerosis and carcinoma cell proliferation5. The rodent homologue of FGF19 is designated FGF15, and because FGF15 and FGF19 activate both human and rodent FGFRs, application of exogenous FGF19 provides a model system to study the effects of exogenous FGF19 on in vivo metabolism in rodents6,7. Interestingly both intraperitoneal injection of FGF19 and overexpression of FGF19 in transgenic mice reversed dietinduced insulin resistance and hyperglycemia in an insulinindependent manner8–10. Recently, Morton et al.1 have reported that a single intracerebroventricular (ICV) injection of FGF19 improved glucose homeostasis in leptin-deficient ob/ob mice without affecting insulin secretion or whole-body insulin sensitivity. This finding, which was independent of changes in body composition or food intake, implies a central action of FGF19 to acutely lower plasma glucose concentrations. In contrast to FGF19, FGF1 is a prototype FGF, which has been well studied for its roles in development, including angiogenesis and morphogenesis11. Surprisingly FGF1 has also recently been shown to lower plasma glucose concentrations in diabetic mice4, while mice genetically deficient in FGF1 exhibit insulin resistance on a high fat diet12. However the molecular mechanism by which FGF1 lowers blood glucose is also unknown. In this regard we have recently demonstrated that increased hypothalamic–pituitary–adrenal (HPA) axis activity, due to acquired leptin deficiency, is critical in promoting hyperglycemia in poorly controlled type 1 diabetes (T1D) and T2D. This study found that increased plasma corticosterone concentrations led to increased rates of lipolysis and gluconeogenesis through increases in the allosteric activation of pyruvate carboxylase (PC) by increases in hepatic acetyl CoA content and increased glycerol turnover13. Similar to the effect of leptin, we hypothesized that ICV administration of FGF1 or FGF19 in a streptozotocininduced rat model of T1D might lower plasma glucose concentrations and normalize rates of hepatic glucose production by suppressing lipolysis through reductions in activation of the HPA axis. Here we show that FGF1 and FGF19 indeed suppress adrenocorticotropic hormone (ACTH) and corticosterone, leading to reductions in whole-body lipolysis, hepatic acetyl CoA content and PC activity, thereby suppressing hepatic glucose production. These data thus identify a common mechanism for the action of FGF1 and FGF19 to ameliorate hyperglycemia in poorly controlled diabetes by suppression of the HPA axis. Results Glucocorticoid suppression reduces lipolysis and HGP. Suppression of glucocorticoid release with ketoconazole resulted 2
in reductions in plasma glucose, non-esterified fatty acid (NEFA) and glycerol concentrations without any change in plasma insulin concentrations (Fig. 1a-d). These reductions in plasma NEFA and glycerol concentrations could be attributed to reductions in whole-body rates of lipolysis as reflected by 60% reductions in rates of glycerol and palmitate turnover and were associated with a similar reduction in hepatic glucose production (Fig. 1e-g). Acetyl CoA, a potent activator of PC14, was also reduced by 50% with ketoconazole treatment (Fig. 1h), again demonstrating a key role for glucocorticoid-induced lipolysis and increased hepatic acetyl CoA in mediating hyperglycemia in a rat model of poorly controlled T1D. FGF19 suppresses HGP by reducing hepatic acetyl CoA. We next examined the hypothesis that ICV injection of FGF19 would improve plasma glucose concentrations in a severely hyperglycemic, insulinopenic rat model of T1D and found that plasma glucose, NEFA and glycerol concentrations were reduced markedly within 6 h following injection of 30 mg ICV FGF19 (Fig. 2a–c). This reduction in hyperglycemia was associated with a 70% reduction in plasma corticosterone and ACTH concentrations but occurred independently of any change in plasma insulin, glucagon, epinephrine, norepinephrine or growth hormone concentrations (Fig. 2d–j). Suppression of corticosterone was associated with reductions in hepatic glucose production and in whole-body lipolysis as reflected by reductions in glycerol and palmitate turnover (Fig. 3a–c). Reduced HGP in FGF19-treated rats was associated with 50–60% reductions in hepatic PC activity as well as hepatic acetyl CoA content (Fig. 3d,e). In contrast this perturbation had no effect on liver (Fig. 3f) or muscle glycogen content (4.5±0.3 versus 4.7±0.2 versus 4.5±0.2 mg g 1), or on hepatic gluconeogenic protein expression (Fig. 3g, Supplementary Fig. 1), Taken together these data suggest that suppression of the HPA axis may contribute to FGF19’s glucose-lowering effect in T1D rats. To further test this hypothesis, we performed a 6-h intraarterial infusion of corticosterone immediately following FGF19 treatment. Restoring plasma corticosterone concentrations in FGF19-treated T1D rats to those measured in untreated T1D rats totally abrogated the glucose-lowering effects of FGF19 as well as its effects on plasma glucose, lactate, NEFA, glycerol concentrations and rates of palmitate and glycerol turnover (Figs 2–3). As predicted by these data, plasma corticosterone concentrations were strong predictors of plasma glucose (r2 ¼ 0.63), palmitate (r2 ¼ 0.81) and glycerol turnover (r2 ¼ 0.84), with both palmitate and glycerol turnover correlating tightly with rates of hepatic glucose production (r2 ¼ 0.60 and 0.61, respectively). To further test the hypothesis that the effects of FGF19 on glycaemia in T1D rodents are due to suppression of the HPA axis and independent of the length of the fasting time (18 h) (Fig. 4a), we repeated these studies in rats with food withdrawn for only 6 h and treated these T1D animals with a three-fold lower ICV dose of FGF19 (10 mg). These studies confirmed the previous observations, with similar reductions in plasma glucose, corticosterone and ACTH concentrations as well as rates of hepatic glucose production, lipolysis and hepatic acetyl CoA content observed in rats treated with low-dose FGF19 and fasted for 6 h. Additionally FGF19 treatment raised plasma lactate concentrations as previously described1 but did not alter rates of wholebody glycolysis. These effects were all abrogated by an intraarterial infusion of corticosterone to match plasma concentrations of untreated T1D rats (Fig. 4b–n). FGF19 suppresses HGP by reducing hepatic acetyl CoA. Next we hypothesized that FGF1 may have similar effects to suppress
NATURE COMMUNICATIONS | 6:6980 | DOI: 10.1038/ncomms7980 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7980
P
DOI: 10.1038/ncomms7980
OPEN
FGF1 and FGF19 reverse diabetes by suppression of the hypothalamic–pituitary–adrenal axis Rachel J. Perry1,2,3, Sangwon Lee2,4, Lie Ma2,4, Dongyan Zhang1, Joseph Schlessinger2,4 & Gerald I. Shulman1,2,3
Fibroblast growth factor-1 (FGF1) and FGF19 have been shown to improve glucose metabolism in diabetic rodents, but how this occurs is unknown. Here to investigate the mechanism of action of these growth factors, we perform intracerebroventricular (ICV) injections of recombinant FGF1 or FGF19 in an awake rat model of type 1 diabetes (T1D) and measure rates of whole-body lipolysis, hepatic acetyl CoA content, pyruvate carboxylase activity and hepatic glucose production. We show that ICV injection of FGF19 or FGF1 leads to a B60% reduction in hepatic glucose production, hepatic acetyl CoA content and whole-body lipolysis, which results from decreases in plasma ACTH and corticosterone concentrations. These effects are abrogated by an intra-arterial infusion of corticosterone. Taken together these studies identify suppression of the HPA axis and ensuing reductions in hepatic acetyl CoA content as a common mechanism responsible for mediating the acute, insulin-independent, glucose-lowering effects of FGF1 and FGF19 in rodents with poorly controlled T1D.
1 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 2 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 3 Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06536-8012, USA. 4 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA. Correspondence and requests for materials should be addressed to G.I.S. (email: [email protected]).
NATURE COMMUNICATIONS | 6:6980 | DOI: 10.1038/ncomms7980 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7980
R
ecent studies have generated a great deal of interest in the insulin-independent glucose-lowering effects of fibroblast growth factor-19 (FGF19) and FGF1 in diabetic rodents and their potential application as novel anti-diabetic therapies1–4; however, little is known regarding the mechanism by which these factors decrease plasma glucose concentrations. FGF19 is an atypical FGF which together with FGF21 and FGF23 is designated an endocrine FGF. It is now well established that members of the FGF family of growth factors mediate their cellular responses by binding together and acting in concert with heparin sulfate proteoglycans to activate the four receptor tyrosine kinases designated FGFR1–4. The endocrine members of the FGF family, designated FGF19, -21 and -23, mediate their endocrine functions by binding selectively to FGFR1c, FGFR2c, FGFR3c or FGFR4 together with b-Klotho or a-Klotho, respectively, to mediate their multiple endocrine functions1–3. FGF19 has been proposed to play diverse physiological roles, modulating development, metabolism, neuronal signalling, atherosclerosis and carcinoma cell proliferation5. The rodent homologue of FGF19 is designated FGF15, and because FGF15 and FGF19 activate both human and rodent FGFRs, application of exogenous FGF19 provides a model system to study the effects of exogenous FGF19 on in vivo metabolism in rodents6,7. Interestingly both intraperitoneal injection of FGF19 and overexpression of FGF19 in transgenic mice reversed dietinduced insulin resistance and hyperglycemia in an insulinindependent manner8–10. Recently, Morton et al.1 have reported that a single intracerebroventricular (ICV) injection of FGF19 improved glucose homeostasis in leptin-deficient ob/ob mice without affecting insulin secretion or whole-body insulin sensitivity. This finding, which was independent of changes in body composition or food intake, implies a central action of FGF19 to acutely lower plasma glucose concentrations. In contrast to FGF19, FGF1 is a prototype FGF, which has been well studied for its roles in development, including angiogenesis and morphogenesis11. Surprisingly FGF1 has also recently been shown to lower plasma glucose concentrations in diabetic mice4, while mice genetically deficient in FGF1 exhibit insulin resistance on a high fat diet12. However the molecular mechanism by which FGF1 lowers blood glucose is also unknown. In this regard we have recently demonstrated that increased hypothalamic–pituitary–adrenal (HPA) axis activity, due to acquired leptin deficiency, is critical in promoting hyperglycemia in poorly controlled type 1 diabetes (T1D) and T2D. This study found that increased plasma corticosterone concentrations led to increased rates of lipolysis and gluconeogenesis through increases in the allosteric activation of pyruvate carboxylase (PC) by increases in hepatic acetyl CoA content and increased glycerol turnover13. Similar to the effect of leptin, we hypothesized that ICV administration of FGF1 or FGF19 in a streptozotocininduced rat model of T1D might lower plasma glucose concentrations and normalize rates of hepatic glucose production by suppressing lipolysis through reductions in activation of the HPA axis. Here we show that FGF1 and FGF19 indeed suppress adrenocorticotropic hormone (ACTH) and corticosterone, leading to reductions in whole-body lipolysis, hepatic acetyl CoA content and PC activity, thereby suppressing hepatic glucose production. These data thus identify a common mechanism for the action of FGF1 and FGF19 to ameliorate hyperglycemia in poorly controlled diabetes by suppression of the HPA axis. Results Glucocorticoid suppression reduces lipolysis and HGP. Suppression of glucocorticoid release with ketoconazole resulted 2
in reductions in plasma glucose, non-esterified fatty acid (NEFA) and glycerol concentrations without any change in plasma insulin concentrations (Fig. 1a-d). These reductions in plasma NEFA and glycerol concentrations could be attributed to reductions in whole-body rates of lipolysis as reflected by 60% reductions in rates of glycerol and palmitate turnover and were associated with a similar reduction in hepatic glucose production (Fig. 1e-g). Acetyl CoA, a potent activator of PC14, was also reduced by 50% with ketoconazole treatment (Fig. 1h), again demonstrating a key role for glucocorticoid-induced lipolysis and increased hepatic acetyl CoA in mediating hyperglycemia in a rat model of poorly controlled T1D. FGF19 suppresses HGP by reducing hepatic acetyl CoA. We next examined the hypothesis that ICV injection of FGF19 would improve plasma glucose concentrations in a severely hyperglycemic, insulinopenic rat model of T1D and found that plasma glucose, NEFA and glycerol concentrations were reduced markedly within 6 h following injection of 30 mg ICV FGF19 (Fig. 2a–c). This reduction in hyperglycemia was associated with a 70% reduction in plasma corticosterone and ACTH concentrations but occurred independently of any change in plasma insulin, glucagon, epinephrine, norepinephrine or growth hormone concentrations (Fig. 2d–j). Suppression of corticosterone was associated with reductions in hepatic glucose production and in whole-body lipolysis as reflected by reductions in glycerol and palmitate turnover (Fig. 3a–c). Reduced HGP in FGF19-treated rats was associated with 50–60% reductions in hepatic PC activity as well as hepatic acetyl CoA content (Fig. 3d,e). In contrast this perturbation had no effect on liver (Fig. 3f) or muscle glycogen content (4.5±0.3 versus 4.7±0.2 versus 4.5±0.2 mg g 1), or on hepatic gluconeogenic protein expression (Fig. 3g, Supplementary Fig. 1), Taken together these data suggest that suppression of the HPA axis may contribute to FGF19’s glucose-lowering effect in T1D rats. To further test this hypothesis, we performed a 6-h intraarterial infusion of corticosterone immediately following FGF19 treatment. Restoring plasma corticosterone concentrations in FGF19-treated T1D rats to those measured in untreated T1D rats totally abrogated the glucose-lowering effects of FGF19 as well as its effects on plasma glucose, lactate, NEFA, glycerol concentrations and rates of palmitate and glycerol turnover (Figs 2–3). As predicted by these data, plasma corticosterone concentrations were strong predictors of plasma glucose (r2 ¼ 0.63), palmitate (r2 ¼ 0.81) and glycerol turnover (r2 ¼ 0.84), with both palmitate and glycerol turnover correlating tightly with rates of hepatic glucose production (r2 ¼ 0.60 and 0.61, respectively). To further test the hypothesis that the effects of FGF19 on glycaemia in T1D rodents are due to suppression of the HPA axis and independent of the length of the fasting time (18 h) (Fig. 4a), we repeated these studies in rats with food withdrawn for only 6 h and treated these T1D animals with a three-fold lower ICV dose of FGF19 (10 mg). These studies confirmed the previous observations, with similar reductions in plasma glucose, corticosterone and ACTH concentrations as well as rates of hepatic glucose production, lipolysis and hepatic acetyl CoA content observed in rats treated with low-dose FGF19 and fasted for 6 h. Additionally FGF19 treatment raised plasma lactate concentrations as previously described1 but did not alter rates of wholebody glycolysis. These effects were all abrogated by an intraarterial infusion of corticosterone to match plasma concentrations of untreated T1D rats (Fig. 4b–n). FGF19 suppresses HGP by reducing hepatic acetyl CoA. Next we hypothesized that FGF1 may have similar effects to suppress
NATURE COMMUNICATIONS | 6:6980 | DOI: 10.1038/ncomms7980 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7980
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